We propose using a combination of mass spectrometry (MS)-based approaches to discover and verify candidate biomarkers of primary Sjgren's Syndrome (pSS) in whole saliva. We are focusing on pSS due to a very large clinical need for a definitive non-invasive assay for diagnosing this debilitating disease. Accordingly, we propose testing the hypothesis that pSS exocrinopathy is accompanied by alterations in the salivary proteome and/or glycome and that MS can be used to identify these disease-related changes. We will utilize whole saliva samples obtained from the Sjgren's International Collaborative Clinical Alliance (SICCA). In recently completed experiments, we compared the composition of saliva samples collected from Sjgren's patients and control individuals. At the protein level, we discovered disease-related alterations in the relative abundances of several components. Additionally, an immunoblotting approach revealed pSS-associated changes in glycosylation. Specifically, in samples from pSS patients, we detected uniformly high levels of the unusual carbohydrate structure, sialyl Lewis x with sulfation on the 6-position of N-acetylglucosamine, which mediates rolling and tethering during leukocyte recruitment from the blood. These findings suggested the existence of disease-related changes in salivary composition that can be exploited as biomarkers. The proposed work includes the discovery and verification phases of the biomarker discovery process with development of a clinical immunoassay and validation of a marker panel as the second stage, i.e., outside the scope of the planned experiments. Specifically, in Aim 1, the discovery phase, we will employ untargeted and targeted MS-based strategies to identify biomarkers of pSS. In the untargeted approach, peptide mixtures that are generated from whole saliva samples of pSS patients and control individuals will be labeled with isobaric mass tags (iTRAQ reagents) and the deep proteome surveyed by MS. In targeted approaches, we will use lectin and antibody affinity chromatography at the glycopeptide level with MS-based sequencing to discover pSS-associated differences in the glycosylation of salivary proteins and to locate the modified sites along the peptide backbone. In Aim 2, the verification phase, we will use immunoassays when possible or, in their absence, multiple-reaction monitoring (MRM) MS to quantify proteins in a larger set of whole saliva samples, also collected by SICCA, from additional pSS patients and control subjects. Thus, at the conclusion of the proposed experiments we will have identified candidate biomarkers of pSS that could lead to a noninvasive clinical test for diagnosing this condition. In addition, the results of these experiments could have other important outcomes. For example, a subset of the biomarkers we discover may also be useful for diagnosing SS in patients who have other autoimmune diseases. Finally, disease-related changes in the composition and/or glycosylation of salivary proteins could give us interesting mechanistic insights into the pathogenesis of this poorly understood condition.